Transport of dopamine (DA) into DA neurons by the plasma membrane DA transporter (DAT) is a critical mechanism controlling the duration and intensity of DA neurotransmission in the brain. Also, drugs like amphetamine and cocaine produce psychomotor stimulation by inhibiting DA uptake by DAT. Since DAT is only active when expressed at the surface of neurons, it is vital to understand how the amount of DAT in the plasma membrane is rapidly regulated by endocytosis and recycling. The mechanisms of these trafficking processes are largely unknown. Therefore, the overall goal of this project is to elucidate how the amount of DAT in the plasma membrane, and thus its activity, are regulated by trafficking of DAT in DA neurons. The data generated in our laboratory and by others suggest that endocytic trafficking of DAT is controlled by unique and novel mechanisms. Our preliminary results suggest that DAT internalization and/or intracellular sorting of internalized DAT is controlled by DAT ubiquitylation. To elucidate the role of DAT ubiquitylation and to define further the mechanisms of DAT endocytosis, a set of novel reagents, viz., extracellularly-tagged DAT and "photoswitchable" DAT fusion proteins, and novel methodologies based on large-scale functional analyses using short interfering RNAs (siRNAs) targeted to trafficking proteins have been developed. These new reagents and methodologies will be used in model cell systems, including co- cultures of rat primary dissociated embryonic mesencephalic (MES)-striatal (STR) neurons. Synaptosomes and slices acutely prepared from adult rat MES or STR will also be used. The specific aims of the proposed work are: (1) to determine the role of DAT ubiquitylation in DAT internalization and post-endocytic sorting;(2) to define the pathways and downstream mechanisms of ubiquitin-dependent and -independent internalization of DAT;and (3) to test the hypothesis that brain-derived neuronal factor (BDNF) activation of the receptor tyrosine kinase TrkB acutely increases DAT activity by reducing endocytosis of DAT and to elucidate the molecular mechanisms underlying this up-regulation. All three aims are interdependent in that the information, new concepts and experimental tools developed in each aim will benefit the research in other aims of this project. The results should provide novel insights about the regulated trafficking of DAT in DA neurons. As DAT trafficking and its regulation are better understood, the role of DAT in normal neurotransmission, as well as its role in brain diseases such as drug addiction, will be better appreciated.